Generation of monodisperse alginate microbeads and <Emphasis Type="Italic">in situ</Emphasis> encapsulation of cell in microfluidic device

A microfluidic method for the in situ production of monodispersed alginate hydrogels using chaotic mixing is described. Aqueous droplets comprising of alginate and calcium as a cross-linking agent were formed as an immiscible continuous phase, and then the alginate and calcium in the droplet came into contact and were rapidly mixed. Gelation of the hydrogel was achieved in situ by the chaotic mixing of the droplets in the microfluidic device. Important operating parameters included: the capillary number (Ca) and the flow rate of the continuous phase, which mainly influenced the formation of three distinctive flow regimes, such as fluctuation, stable droplets, and laminar flow. Under the stable formation of droplets regime, monodispersed alginate microbeads having a narrow size distribution (below 3% of CV) were produced in the microfluidic device and the size of the microbeads, ranging from 60 to 95 μm, could be easily modulated by varying the flow rate, viscosity, and interfacial tension. In addition, this approach can be applied to the encapsulation of yeast cells in alginate hydrogels with a high monodispersity. This simple microfluidic technique for the production of monodispersed hydrogels and encapsulation of biomolecules shows strong potential for use in biosensors, cell sensors, drug delivery systems, and cell transplantation applications.     

Islet microencapsulation: a review

The original report on the microencapsulation of islets of Langerhans used sodium alginate and poly-l-lysine (PLL) to form the capsules. Although several alternative materials have subsequently been used with varying degrees of success, it is those studies using islets encapsulated in alginate-PLL-alginate which are reviewed in detail in this article. Since the first report of islet microencapsulation, many studies have demonstrated excellent in vitro viability of encapsulated islets. However, transplantation experiments into chemically induced diabetic recipients have yielded varied results, with some studies showing good long-term graft function whilst in others grafts failed due to pericapsular fibrosis. The use of naturally occurring animal models of type 1 (insulin-dependent) diabetes has demonstrated a decline in graft function, suggesting that this presents a more complex problem to be solved than that in chemically induced diabetic recipients. Fibrosis of capsules has been the major problem causing graft failure, and this has been demonstrated to be more severe in spontaneously diabetic models. However, recent advances in alginate purification and attempts to reduce the size of the encapsulated islets are major steps towards encapsulated islet transplants becoming a viable proposition for the treatment of type 1 diabetic patients.     

Culture and differentiation of chondrocytes entrapped in alginate gels

Summary We studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.     

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-γ cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Often the transfected cells are xenogeneic or allogeneic to the experimental animal and have to be encapsulated in such a way that no cellular response by the host will be induced. Alginate has proven to be a simple matrix for encapsulating cells under mild conditions suitable for in vivo implantation. Encapsulated cells express the transfected IL-4 gene for at least 14 days after in vivo implantation and were shown to be functional during that period by modulating ongoing IgE responses. The application of adherent growing transfected cells permits dose-response titrations and provides an easy method for local and systemic cytokine delivery. Alternatively, hybridoma cells can be encapsulated and the secreted antibody monitored in the serum. It was found that no host immune response was triggered by alginate encapsulated cells. The efficiency of treatment by encapsulated cells was shown to be equivalent to that of injecting purified antibodies.     

Effect of additives on the viability of bifidobacteria loaded in alginate poly-l-lysine microparticles during the freeze-drying process

Bifidobacteria-loaded alginate poly-l-lysine microparticles (bap microparticles) were prepared using an air atomization method and then freeze-dried. The viability of the bap microparticles was investigated as a function of the amount of the bifidobacteria cultures, and the addition of a yeast extract, cryoprotectants, antioxidants and neutralizer. The size of the bap microparticles with and without the bifidobacteria was 84.8 +/- 28.5 microm (mean +/- standard deviation) and 113.1 +/- 38.5 microm, respectively. The surface morphology was slightly ellipsoid and wrinkled regardless of the incorporating bifidobacteria. The viability gradually decreased with increasing freeze-drying time. Free-flowing powdered bap microparticles were obtained at least 12 h after freeze-drying the wetted slurry of bap microparticles. However, the particles tended to aggregate when either lactose or ascorbic acid was added. The addition of a yeast extract, cryoprotectants (glycerol and lactose), antioxidants (NaHSO3 and ascorbic acid) and neutralizer (Mg3(PO4)2) resulted in a significantly higher viability of the bifidobacteria in the bap microparticles after freeze-drying (0.34-1.84 log) compared with the culture alone.     

The use of a dual PEDOT and RGD-functionalized alginate hydrogel coating to provide sustained drug delivery and improved cochlear implant function

Cochlear implants provide hearing by electrically stimulating the auditory nerve. Implant function can be hindered by device design variables, including electrode size and electrode-to-nerve distance, and cochlear environment variables, including the degeneration of the auditory nerve following hair cell loss. We have developed a dual-component cochlear implant coating to improve both the electrical function of the implant and the biological stability of the inner ear, thereby facilitating the long-term perception of sound through a cochlear implant. This coating is a combination of an arginine-glycine-aspartic acid (RGD)-functionalized alginate hydrogel and the conducting polymer poly(3, 4-ethylenedioxythiophene) (PEDOT). Both in vitro and in vivo assays on the effects of these electrode coatings demonstrated improvements in device performance. We found that the coating reduced electrode impedance, improved charge delivery, and locally released significant levels of a trophic factor into cochlear fluids. This coating is non-cytotoxic, clinically relevant, and has the potential to significantly improve the cochlear implant user’s experience.     

Effects of double encapsulation and coating on synthetic seed conversion in M.26 apple rootstock

Encapsulated vitro -derived apical buds of M.26 apple rootstock (Malus pumila Mill) can be employed for the formation of the synthetic seed. Satisfactory levels of conversion (plantlets from synthetic seed) can be achieved if there are adequate (i) rooting induction treatment, (ii) protocol of encapsulation, and (iii) nutritive and environmental conditions. For capsule manufacturing, sodium alginate is largely used; however, this is excessively permeable with loss of the nutritive substances (artificial endosperm) and/or dehydration risks during conservation and transport causing detrimental effects on the synthetic seed conversion and on the plantlet's growth. In order to overcome these problems, two experiments were carried out comparing simple encapsulation in alginate with double encapsulation, and with encapsulation-coating procedures. The presence of a second layer of alginate (double encapsulation) and of a thin external coating layer over the alginate (encapsulation-coating) did not show any detrimental effects on viability, sprouting and regrowth of the encapsulated microcuttings. Satisfactory conversion (70%) was reached with the encapsulation-coating procedure, whereas the double and simple encapsulation converted less than 40% of the synthetic seed. The effect of the addition to the capsule of an anti-microbial substance (Plant Preservative Mixture - PPM#174;) was examined: it did not compromise the conversion of the encapsulated microcuttings sown in ex-vitro non-aseptic conditions.     

Quantitative study of the production and properties of alginate/poly-L-lysine microcapsules

Alginate-polylysine-alginate (APA) microcapsules are of particular interest for their application as implants or for bioreactor cultures. Although their formation has been widely studied, there is still a lack of quantitative data describing resistance, membrance thickness and permeability. In this study, the quantitative application of a Texture Analyser for the measurement of capsule deformation yielded important results that permit comparison with other polymer systems used for encapsulation. Furthermore, single-membrane and multi-membrane capsules were formed in order to improve the modulation of the capsule properties. For single-membrane capsules, resistance was mostly affected by the incubation time in poly-L-lysine (PLL), the PLL molecular weight and concentration. The increase in resistance from 0.1 #45 0.01 g/capsules to 2 #45 0.2 g/capsules was linked to a membrane thickening (35-120 #181;m) and a decrease in permeability (150 to 40 kD). Thus, it was not possible to modify resistance and membrane permeability independently. Multi-membrane capsules with a resistance comparable to single-membrane capsules could be formed using various combinations of PLL molecular weights, and enabled uncoupling of permeability and resistance properties.     

Radiolabeling and evaluation of alginate blend-isoniazid microspheres by 99mTc for the treatment of tuberculosis in rabbit model

Isoniazid (INH) is the first line anti-tubercular drug that is widely used in the treatment of tuberculosis. ^99mTc-alginate-INH microsphere scintigraphy has been demonstrated to be a useful noninvasive imaging technique for microsphere deposits located in different organs of the rabbits. The aim of this study was to develop an improved formulation, to validate the formulation for long-time retention, as well as to assess radiotracer stability by novel quality control methods. Our study reports the labeling and evaluation of alginate blends-INH microspheres. The incorporation efficiency of optimized formulation was 89% w/w. The in vitro release study was carried out in simulated intestinal fluid at pH 7.4, and it was found that the formulation delivered the drug for 36 h. The labeling efficiency of ^99mTc-alginate blends-INH microspheres was seen at various pH (i.e. pH ranging from 5 to 7.5) and different concentration of stannous chloride dehydrate (i.e. 25–200 μg) and it was concluded that 96% labeling efficiency was achieved in case of pH 7.5 and 60 μg stannous chloride. The stability study was carried out in saline and serum and it was found that the complex was highly stable in vitro and in vivo. The blood clearance in rabbits showed bi-exponential pattern depicting that 50% of activity washed out at 2 h with t_1/2(Fast) was 2.1 h and t_1/2(Slow) was 12.5 h. Bio-distribution was normal and the experimental mice showed major accumulation of the radiolabeled formulation in liver, intestine, lungs and kidneys, indicating hepatobiliary and renal route of excretion. The distribution of the drugs to the lung was showing its efficiency in the treatment of tuberculosis.     

Bioefficacy of budesonide loaded crosslinked polyeletrolyte microparticles in rat model of induced colitis

A targeted delivery system for inflammatory bowel diseases, chitosan-Ca-alginate microparticles efficiently loaded with budesonide (BDS), were designed using one-step spray-drying process. They were eudragit-coated and examined for in vivo efficacy. Experimental colitis was induced by rectal instillation of 2,4,6-trinitrobenzene sulphonic acid (TNBS) into male Wistar rats. Drugs were administered by oral gavage daily for 5 days. Colon/body weight ratio, gross morphological and histological evaluation, and clinical activity score were determined as inflammatory indices. Individual clinical and histological evaluation showed that colitis severity was suppressed the most greatly in order BDS < BDS/C-Ca-A < E-BDS/C-Ca-A. Clinical activity score decreased in the same order. Statistical analyses of total score points indicate that the incorporation of BDS in microparticles had significant differences in favor of efficacy of designed delivery system with mucoadhesive and controlled release properties (one-way ANOVA, P?<?0.05). The results established the prediction by previous in vitro studies.